Comparative Biochemistry and Physiology Part D: Genomics and Proteomics
Proteomic analysis of extracellular medium of cryopreserved carp (Cyprinus carpio L.) semen
Graphical abstract
Introduction
Cryopreservation of fish sperm is an important tool for the conservation of biodiversity (fish sperm cryobanks) and the efficient and selective fertilization and synchronization of artificial reproduction (Zilli et al., 2014). The common carp is one of the most important farmed freshwater fish species in the world, and, besides being of commercial interest, it is also a research model organism within Teleostei. As one of the first domesticated fish (Balon, 1995), carp have undergone selective breeding, resulting in different strains. Cryopreservation can be used for securing the sperm of desired strains of common carp or koi carp in sperm banks and for the transportation of semen (Lubzens et al., 1993, McAndrew et al., 1995). Moreover, cryopreservation techniques ensure the availability of sperm through the entire year, enabling cross fertilization between strains or related species that have a distinct period of maturation (for example it is possible to collect sperm in March and fertilize the eggs from species or strains which spawn in June). Although cryopreservation protocols of carp spermatozoa have been established (Gwo et al., 1993, Linhart et al., 2000, Horváth et al., 2003, Warnecke and Pluta, 2003), a decrease of viability and motility of sperm is still observed in cryopreserved semen. For this reason, to our knowledge, cryopreservation has not been implemented into breeding programs for carp.
The decrease in sperm quality after cryopreservation could be attributed to irreversible sublethal cryodamage, which occurs during freezing and thawing. The cryodamage to sperm structure and function may be produced directly by ice formation or by oxidative stress and high osmotic pressure (Li et al., 2010a). Cryopreservation led to alteration in DNA and protein integrity (Labbe et al., 2001, Zilli et al., 2003, Zilli et al., 2005), membrane lipids (Müller et al., 2008), sperm motility (Linhart et al., 2000) and directly engaged cell ability for successful fertilization and embryonic development (Suquet et al., 1998, Kopeika et al., 2003). The changes in sperm membrane composition and lipid location after cryopreservation have a deleterious effect on spermatozoa plasma membrane integrity (Schuffner et al., 2001). This sperm membrane damage leads to a leakage of the intracellular components of spermatozoa, including cytoplasmic and membrane bound proteins and enzymes as well as other components which co-elute from spermatozoa leading to reduced metabolic activity and, consequently, to a decrease in sperm quality (Gadea et al., 2004, Li et al., 2010b).
Recent studies demonstrated that the loss of spermatozoa functions could also be attributed to the effect of cryopreservation on sperm proteins (Gadea et al., 2004, Zilli et al., 2005, Zilli et al., 2014, Wang et al., 2013). Sperm protein loss can be partially responsible for a decrease in sperm quality after cryopreservation. To our knowledge, there is limited information about changes in the proteome profile in fish sperm that occurs due to cryostorage. So far, only a limited number of proteins (3–12 protein spots), which are affected by cryopreservation, have been identified, in sea bass and carp spermatozoa (Zilli et al., 2005, Zilli et al., 2014, Li et al., 2010b). In these studies, traditional 2DE analysis was used to determine changes in spermatozoa due to cryopreservation. In most cases, a decrease in sperm protein concentration after cryopreservation was recorded. In the present study, we monitored proteins released from spermatozoa by the analysis of an extracellular medium after cryopreservation. By using this approach, it is possible to identify more proteins released from spermatozoa.
The aim of our work was to evaluate the changes in protein composition in the extracellular medium using two independent strategies: 2D-DIGE and 1D-SDS-PAGE combined with LC–MS/MS. The highlighted proteins that were affected by long-term cold storage might be viable biomarkers of cryodamage and will form a useful starting point for future studies. We believe that our findings are the first step in a targeted and detailed analysis of cryoinjuries to carp spermatozoa.
Section snippets
Gamete collection
Milt of common carp was obtained from fish maintained at the Institute of Ichthyobiology and Aquaculture of the Polish Academy of Sciences in Gołysz, Poland. Twenty-four hours before the collection of carp semen, the males were injected intradorsaly with Ovopel (one pellet containing of 18–20 μg of a GnRH analog and 8–10 mg of metoclopramide per 1 kg of fish bw; Interfish Ltd, Hungary). The milt samples were collected by gentle abdominal massage, taking care not to pollute them with blood, feces
2D-DIGE comparison of the extracellular fluid of fresh semen and the extracellular medium of cryopreserved semen
The quantitative comparison of proteome profiles between EF and EM using 2D-DIGE yielded 116 protein spots (ratio > 2, p < 0.01 with FDR correction), and the spots were significantly more abundant in EM. Fig. 1 shows representative 2D-DIGE images as examples for the gel quality and the sample complexity of the overall gel set. We marked the spots (1–116) with a higher intensity in EM in Fig. 1B and the corresponding spots in EF in Fig. 1A. The overlay of EF and EM is shown in Fig. 1C. To illustrate
Discussion
We present the first in-depth proteomic analysis of the extracellular medium obtained after cryopreservation of carp semen. Until recently, the study of fish sperm cryoinjuries was based on the analysis of a few enzymes, such as LDH, malate dehydrogenase, isocitrate dehydrogenase and β-d-glucuronidase (Nynca et al., 2012, Zilli and Vilella, 2012), which leak from sperm to the extracellular medium. Only restricted numbers of sperm proteins (3–10 proteins) affected by cryopreservation in sea bass
Acknowledgment
We thank Florian Flenkenthaler, Daniela Deutsch, Miwako Kösters and Ewa Liszewska for their excellent technical assistance. The authors would like to thank the two anonymous reviewers for their valuable comments and suggestions. This work was supported by Project 2011/01/D/NZ9/00628 from the National Science Centre (Identification and characterization of specific carp seminal plasma proteins — proteomics and a classical approach), funds appropriated to the Institute of Animal Reproduction and
References (45)
- et al.
Sperm motility in fishes. (II) Effects of ions and molality: a review
Cell Biol. Int.
(2006) - et al.
Human sperm tail proteome suggests new endogenous metabolic pathways
Mol. Cell. Proteomics
(2013) Origin and domestication of the wild carp, Cyprinus carpio: from Roman gourmets to the swimming flowers
Aquaculture
(1995)- et al.
Evaluation of DNA damage in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) cryopreserved sperm
Cryobiology
(2005) - et al.
Estimation of sperm concentration of rainbow trout, whitefish and yellow perch using spectrophotometric technique
Aquaculture
(1993) - et al.
Effect of cryopreservation and theophylline on motility characteristics of lake sturgeon (Acipenser fulvescens) spermatozoa
Theriogenology
(1996) - et al.
Characterization of carp seminal plasma proteome in relation to blood plasma
J. Proteomics
(2014) - et al.
In-depth proteomic analysis of carp (Cyprinus carpio L) spermatozoa
Comp. Biochem. Physiol. Part D Genomics Proteomics
(2014) - et al.
Decrease in glutathione content in boar sperm after cryopreservation—effect of the addition of reduced glutathione to the freezing and thawing extenders
Theriogenology
(2004) - et al.
Cryopreservation of common carp sperm
Aquat. Living Resour.
(2003)
Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa
Theriogenology
Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid
Dev. Biol.
Detrimental effects of cryopreservation of loach (Misgurnus fossilis) sperm on subsequent embryo development are reversed by incubating fertilised eggs in caffeine
Cryobiology
Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L.
Theriogenology
Cryopreservation of sperm in common carp Cyprinus carpio: sperm motility and hatching success of embryos
Cryobiology
Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus)
Theriogenology
Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility
Comp. Biochem. Physiol.
Long-term effects of the cryopreservation of turbot (Psetta maxima) spermatozoa
Aquat. Living Resour.
In-depth proteomic analysis of the human sperm reveals complex protein compositions
J. Proteomics
Motility and fertilizing capacity of frozen/thawed common carp (Cyprinus carpio L.) sperm using dimethyl-acetamide as the main cryoprotectant
Aquaculture
Polymorphism of transferrin of carp seminal plasma: relationship to blood transferrin and sperm motility characteristics
Comp. Biochem. Physiol. B
Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation
Cryobiology
Cited by (28)
Effects of activation and assisted reproduction techniques on the composition, structure, and properties of the sauger (Sander Canadensis) spermatozoa plasma membrane
2023, TheriogenologyCitation Excerpt :Cryopreservation is associated with widespread plasma membrane damage in nearly every species. Changes to membrane composition [23,24], fluidity [25], lipid raft integrity [4,26–28] and structural integrity [29] are all implicated in reduced fertilization potential observed in frozen sperm [30]. By contrast, testicular harvest affects sperm less adversely than cryopreservation [31,32] but can still lower fertilization potential in some species [33–35].
Sublethal sperm freezing damage: Manifestations and solutions
2018, Theriogenology